effects of selected additives for growth efficiency...

EFFECTS OF SELECTED ADDITIVES FOR GROWTH

EFFICIENCY ON in vitro GROWN

Vanilla planifolia Andrews

GOH FOONG JING

UNIVERSITI SAINS MALAYSIA

2016

EFFECTS OF SELECTED ADDITIVES FOR GROWTH

EFFICIENCY ON in vitro GROWN

Vanilla planifolia Andrews

by

GOH FOONG JING

Thesis submitted in fulfilment of the requirements for the degree of

Master of Science

March 2016

ii

ACKNOWLEDGEMENT

First of all, I would like to thank to my supervisor, Associate Professor Dr.

Sreeramanan Subramaniam for giving me the invaluable opportunities, patience and

guidance through this research project. I would also like to apologize for the trouble

that I caused. I would like to thank my co-supervisors, Dr. Chew Bee Lynn and

Dr .Rahmad Zakaria from Universiti Sains Malaysia for all their guidelines and

supports. A thank also to Dr. Zuraida Abdul Rahman from Malaysian Agricultural

Research and Development Institute (MARDI) as my field supervisor for all her plant

materials, guidelines and supports.

For all friends of Plant Biotechnology Laboratory 310, Universiti Sains

Malaysia, without your help and friendship, I would go through this journey differently

and would not be completed without your help. Credits go to Safiah, Arulville,

Pavalakodi, Jessica, Monica, Jasim, Suhana, Masyithah, Raheleh, Ranjetta, Soo Ping,

Chee Keong, Winson, Qian Yi, and Ze Hong. Very special thanks to all laboratory

assistants of School of Biological Sciences for providing their assistance in this project.

Very sincere thanks and apologizes goes to my dear parents; Goh Lim Chua

and Liew Swee Chin, my brother and sister for their support, encouragement, patience

and understand. Finally, thanks to all my friends in Universiti Sains Malaysia for all

the memories we shared.

Thanks a lot and wish you all happy and healthy always.

GOH FOONG JING.

iii

TABLES OF CONTENTS

Page

ACKNOWLEDGMENT

ii

TABLES OF CONTENTS

iii

LIST OF TABLES

viii

LIST OF FIGURES

x

LIST OF PLATES

xi

LIST OF ACRONYMS AND ABBREVIATIONS

xiii

ABSTRAK

xv

ABSTRACT

xvii

CHAPTER 1

INTRODUCTION 1

1.1 Objectives 4

CHAPTER 2

LITERITURE REVIEW 5

2.1 Orchids 5

2.1.1 Vanilla genus 5

2.1.2 Vanilla planifolia Andrews’ history and

distribution

6

2.1.3 Vanillin and its uses 7

2.2 Plant tissue culture 10

2.2.1 Orchid micropropagation 11

2.2.2 Culture medium 12

2.2.3 Gelling agent 14

2.2.4 Cytokinin 16

2.2.5 Organic additives 16

2.2.5.1 Coconut water 17

2.2.5.2 Coconut sugar 18

iv

2.2.5.3 Activated charcoal 18

2.2.5.4 Pineapple juice 19

2.2.5.5 Dragon fruit (pitaya) juice 20

2.2.5.6 Banana pulp 21

2.2.5.7 Green tea leaf extract 22

2.2.6 Rooting 22

2.2.7 Acclimatization 23

2.3 Histological analysis

24

CHAPTER 3

MATERIALS AND METHODS 26

3.1 In vitro propagation of Vanilla planifolia Andrews 26

3.1.1 Effects of different strengths of MS medium 27

3.1.2 Effects of different concentrations of BAP 27

3.1.3 Effects of different sucrose concentrations 27

3.1.4 Effects of different gelling agents 27

3.2 Effects of organic additives 28

3.2.1 Coconut water 28

3.2.2 Pineapple juice 29

3.2.3 Banana pulp 29

3.2.4 Dragon fruits juice 30

3.2.5 Activated charcoal 30

3.2.6 Coconut sugar 31

3.2.7 Tea leaf extract

31

3.2.8 Effects of different organic additives

31

3.3 Multiple shoots formation with organic additives 32

3.4 Rooting 32

v

3.5 Acclimatization 32

3.6 Histology and scanning electron microscopy analyses

of in vitro plantlets and ex vitro plants.

33

3.6.1 Paraffin wax embedding in histological

analysis

33

3.6.2 Fresh samples histological analysis 35

3.6.3 Scanning electron microscope analysis 36

3.7 Statistical analysis 36

CHAPTER 4

RESULTS 37


4.1.1 Effects of different strengths of MS basal

medium

37




4.2 Effects of organic additives 49







4.2.7 Tea leaf extract 67

4.2.8 Effects of different organic additives 70


4.4 Rooting 77


vi



81

4.6.1 Paraffin wax embedding in histological

analysis

81

4.6.2 Fresh samples histological analysis 85

4.6.3 Scanning electron microscope analysis 89

CHAPTER 5

DISCUSSION 93


5.1.1 Effects of different strengths of MS basal

medium

93




5.2 Effect of organic additives 97







5.2.7 Tea leaf extract 103

5.2.8 Effects of different organic additives 105


5.4 Rooting 106




108

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CHAPTER 6

CONCLUSION AND FUTURE WORK 111

6.1 Conclusions 111

6.2 Recommendations for future research 112

REFFERENCES

113

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LIST OF TABLES

Page

Table 4.1 Effect of different strengths of MS medium salt (¼, ½, ¾, and

full strength) on the total mean of leaves number (LN), leaves

length (LL), shoot number (SN), plant height (PH), root

number (RN) and root length (RL) of V. planifolia.

42

Table 4.2 Effect of different concentrations of BAP (0, 0.5, 1.0, 2.0 and

4.0 mg/L) supplemented in MS medium on the total mean of

leaves number (LN), leaves length (LL), shoot number (SN),

plant height (PH), root number (RN) and root length (RL) of V.

planifolia.

45

Table 4.3 Effect of different concentrations of sucrose (10, 20, 30 and 40

g/L) supplemented in MS medium on the total mean of leaves

number (LN), leaves length (LL), shoot number (SN), plant

height (PH), root number (RN) and root length (RL) of V.

planifolia.

48

Table 4.4 Effect of different gelling agents (Gelrite, agar, agar-agar and

Isabgol) supplemented in MS medium on total mean of leaves

number (LN), leaves length (LL), shoot number (SN), plant

height (PH), root number (RN) and root length (RL) of V.

planifolia.

51

Table 4.5 Effect of different concentrations of coconut water (0, 10, 20,

30, and 40%) supplemented in MS medium on the total mean

of leaves number (LN), leaves length (LL), shoot number (SN),


planifolia.

54

Table 4.6 Effect of different concentrations of pineapple juice (0, 10, 20,

30 and 40) supplemented in MS medium on the total mean of



planifolia.

57

Table 4.7 Effect of different concentrations of banana pulp (0, 5, 10, 15

and 20 g/L) supplemented in MS medium on the total mean of



planifolia.

60

ix

Table 4.8 Effect of different concentrations of dragon fruit juice (0, 10,

20, 30 and 40 %)supplemented in MS medium on the total

mean of leaves number (LN), leaves length (LL), shoot number

(SN), plant height (PH), root number (RN) and root length

(RL) of V. planifolia.

63

Table 4.9 Effect of different concentrations of activated charcoal (0, 1, 2,

3, and 4 g/L) supplemented in MS medium on the total mean of



planifolia.

66

Table 4.10 Effect of different concentrations of coconut sugar (0, 1, 2, 3,

and 4%) supplemented in MS medium on the total mean of



planifolia.

69

Table 4.11 Effect of different concentrations of tea leaf extract (0, 1, 2, 3,

and 4 g/L) supplemented in MS medium on the total mean of



planifolia.

72

Table 4.12 Effect of selected organic additives supplemented in MS

medium on the total mean of leaves number (LN), leaves length

(LL), shoot number (SN), plant height (PH), root number (RN)

and root length (RL) of V. planifolia.

76

Table 4.13 Effect of different potting medium on leaves number (LN),

leaves length (LL), plant height (PH), aerial root number (RN),

aerial root length (RL) and survival rate (SR) of V. planifolia.

82

x

LIST OF FIGURE

Page

Figure 2.1 Vanillin compound structure

10

Figure 4.1 Mean total number of shoots formed from shoot segments of

V. planifolia on ½ strength MS medium supplemented with

different organic additive combined with BAP after eight

weeks of culture.

79

xi

LIST OF PLATE

Page

Plate 4.1 Development of V. planifolia shoot explants in different

strengths of MS basal medium after 8 weeks of culture.

43


concentration of BAP after eight weeks of culture.

46


concentrations of sucrose after eight weeks of culture.

49

Plate 4.4 Development of V. planifolia cultured in MS medium with

different gelling agents after eight weeks of culture.

52

Plate 4.5 Development of V. planifolia shoot explants in MS medium

supplemented with different concentration of coconut water

after eight weeks of culture.

55


supplemented with different concentration of pineapple juice


58


supplemented with different concentration of banana pulp after

eight weeks of culture.

61


supplemented with different concentration of dragon fruit juice


64


supplemented with different concentration of activated charcoal


67


supplemented with different concentration of coconut sugar


70


supplemented with different concentration of tea leaf extract


73


supplemented with different type of organic additive after eight

weeks of culture.

77

xii


supplemented with different type of organic additive after eight

weeks of culture.

80

Plate 4.14 Acclimatization of V. planifolia plantlets in different potting

medium after 12 weeks of culture.

83

Plate 4.15 V. planifolia plant after 3 months transplanted in the field (bar =

5 cm).

84

Plate 4.16 Histological analysis of plant leaf cross section of in vitro and

ex vitro V. planifolia plant.

86

Plate 4.17 Histological analysis of plant stem cross section of in vitro and


87

Plate 4.18 Histological analysis of plant root cross section of in vitro and


88

Plate 4.19 Fresh sample histological analysis of plant leaf cross section of

in vitro and ex vitro V. planifolia plants.

90

Plate 4.20 Fresh sample histological analysis of plant stem cross section of


91

Plate 4.21 Fresh sample histological analysis of plant stem cross section of


92

Plate 4.22 The morphology of stomata in V. planifolia.

94

Plate 4.23 The morphology of shoot and raphides structures in V.

planifolia.

95

Plate 4.24 The morphology of root, root hairs and root cell structures in V.

planifolia.

96

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LIST OF ACRONYMS AND ABBREVIATIONS

℃ Degree Celsius

$/kg Dollar per kilogram

% Percent/percentage

2iP 6-γ-γ-dimethylaminopurine

ANOVA Analysis of variance

BAP 6-benzylaminopurine

c Chloroplast

cm Centimeter

CRD Completely random design

CW Coconut water

ed Endodermis layer

EDTA Ethlenediaminetetra acetic acid

ep Epidemic layer

FAA Formaliine-acetic acid-alcohol

FESEM Field emission scanning electron microscope

g/L Gram per litre

HCL Hydrochloric acid

IAA Indole-3-acetic acid

IBA Indole-3-butyric acid

kgf/cm2 Kilogram-force/square centimetre

LL Leaves length

LN Leaves number

M Molar

mg/L Milligram per litre

xiv

MS Murashige and Skoog

n Nucleus

N69 Nitsch basal medium

NAA Naphthalene acetic acid

NaOH Sodium hydroxide

N Normality

nm Nano metre

p Phloem

PH Plant height

PLBs Protocorms-like bodies

r Raphides

rh Root hair

RL Root length

RN Root number

SEM Scanning electron microscope

SN Shoot number

st Sieve tube

TBA Tertiary-butyl alcohol

v Vein

vb Vascular bundle

v/v Volume/volume

xv

KESAN BAHAN TAMBAHAN TERPILIH UNTUK KEBERKESANAN

PERTUMBUHAN Vanilla planifolia Andrews YANG TUMBUH SECARA in

vitro

ABSTRAK

Vanilla planifolia Andrews ialah ramuan makanan yang mempunyai nilai

ekonomi dan kedua paling mahal di dunia. Dalam kajian yang lepas, bahan

tambahan organik telah menujuk potensi mengingkatkan perkembangan dan

pertumbuhan pokok tumbuhan tetapi kajian yang teliti terhadap V. planifolia terhad.

Maka, dalam kajian ini, kesan bahan tambahan organik berbeza yang dipilih telah

dikaji terhadap pertumbuhan dan perkembangan V. planifolia Andrews. Peningkatan

protokol yang lebih baik untuk pertumbuhan tumbuhan dan perkembangan vanila

telah dijalankan dan akhirnya mengkaji perbezaan morfologi anak pokok yang

ditanam secara in vitro dan ex vitro. Pucuk V. planifolia dikultur dalam kekuatan

medium MS yang berbeza, kepekatan BAP dan sukrosa yang berbeza, dan jenis ejen

gel yang berlainan untuk mengkaji medium pertumbuhan yang terbaik. Pertumbuhan

anak pokok V. planifolia direkodkan selepas dirawat selama lapan minggu. Hasil

Kajian menunjukkan pembekalan ½ kekuatan medium MS dan 20 g/L sukrosa

didapati cukup berkesan untuk menggalakkan pertumbuhan pokok. Penambahan 1.0

mg/L BAP dapat meningkatkan purata 4.11 ± 0.36 pucuk setiap eksplan. Keempat-

empat jenis ejen gel tidak menunjukkan kesan yang signifikan terhadap pertumbuhan

V. planifolia. Sebanyak tujuh jenis bahan tambahan organik telah dipilih untuk

mengkaji kesan setiap bahan tambahan organik terhadap pertumbuhan dan

perkembangan pucuk eskplan. Penambahan 10% air kelapa ke dalam medium itu

didapati paling berkesan untuk meningkatkan pertumbuhan pokok V. Planifolia.

Dengan penambahan 30% air kelapa atau 2 g/L ekstrak daun teh hijau, penggandaan

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anak pokok V. Planifolia menghasilkan keputusan yang agak sama dengan 4.26 ±

0.43 (air kelapa) dan 4.00 ± 0.86 (ekstrak daun teh hijau) pucuk setiap eksplan

berbanding dengan medium yang ditambah dengan 1 mg/L BAP. Walau

bagaimanapun, kombinasi 1 mg/L BAP dan 10 % air kelapa dapat meningkatkan

penghasilan pucuk baru sebanyak 30%. Pertumbuhan akar V. planifolia dapat

memcapai 100% tanpa penambahan hormon dan bahan tambahan organik. Lumut

sphagnum didapati merupakan medium berpasu yang paling sesuai untuk aklimitasi

V. planifolia dengan pencapai kadar kemandiran 83.33 %. Histologi dan imbasan

mikroskop elektron telah digunakan untuk mengkaji perubahan morfologi V.

planifolia di bahagian daun, batang dan akar yang ditanam secara in vitro dan ex

vitro. Bedasarkan kajian histologi, perbezaan struktur dalam anak pokok in vitro dan

anak pokok ex vitro telah ditunjuk. SEM menunjukkan stomata anak pokok in vitro

yang kurang lengkap dan kewujudan lilin epikutikular disekeliling stomata. SEM

juga menunjukkan kewujudan raphid dalam V. planifolia. Kajian ini telah

menunjukan potensi bahan tambahan organik yang kurang didedahkan sebagai

penyelesaian alternatif untuk meningkatkan pertumbuhan dan perkembangan pokok

serta penyesuaian morfologi daripada keadaan in vitro terhadap keadaan ex vitro

demi kepentingan kemandirian pokok.

xvii

EFFECTS OF SELECTED ADDITIVES FOR GROWTH EFFICIENCY ON in

vitro GROWN Vanilla planifolia Andrews

ABSTRACT

Vanilla planifolia Andrews is the economically important and second most

expensive food ingredients in the worldwide. In previous studies, organic additives

have shown their potential on promoting plant growth and development, however the

detail studies of organic additive on plant growth and development of V. planifolia

was limited. Thus, in this research, the effects of different selected organic additives

were studied on the plant growth and development of V. planifolia Andrews.

Improvement on the protocol for plant growth and proliferation of vanilla were

carried out and finally to study the morphological differences between the in vitro

and ex vitro plantlets. V. planifolia plant shoots were initially cultured on different

strengths of MS, different concentrations of BAP, sucrose and different types of

gelling to determine the best growth medium. The V. planifolia plantlets growth

were recorded after eight weeks of treatment. Supplemented of ½ strength MS

medium and supplementation of 20 g/L were efficient enough to promoted plant

growth. Addition of 1.0 mg/L BAP was found to induce an average of 4.11 ± 0.36

shoots per explant. The four gelling agents used were found to have no significant

effect on the plant growth of V. planifolia. There were seven types of organic

additives were selected to study the effect of each additive towards the plant growth

and development of the shoots explant. Inclusion of 10% coconut water into the

medium was found to be the most effective for enhancement of V. planifolia plant

growth. With an addition of 30% coconut water or tea leaf extract, the multiplication

of V. planifolia plantlets was found to produce similar results with 4.26 ± 0.43

(coconut water) and 4.00 ± 0.86 (tea leaf extract) shoots per explant as compared to

xviii

the basal medium with addition of 1 mg/L BAP. However, with the combinations of

1mg/L of BAP and 10% of coconut water, multiple shoots formation was able to

increase about 30%. V. planifolia was able to perform 100% rooting without any

plant growth regulator and organic additive. Sphagnum moss was found to be the

more suitable potting medium as the survival rate of acclimatization of V. planifolia

were able to archived 83.33% of survival rates. Histological studies and scanning

electron microscopy were carried out to determine the morphological changes of V.

planifolia on both in vitro and ex vitro plant leaves, stems and roots of V. planifolia.

According to the histological analyses, there were structure differences in in vitro

plantlets and ex vitro plants. Scanning electron micrograph displayed the under-

develop stomata of plantlets and existent of epicuticular wax surrounding the stomata.

SEM also displayed the existent of raphides in V. planifolia. This study had shown

the potential of organic additive that are not fully discovered as an alternative

solution for enhancement of plant growth and development and also the

morphological adaptation changes from in vitro condition to ex vitro condition which

is vital for plant survival.

1

CHAPTER 1

INTRODUCTION

Vanilla (Vanilla planifolia Andrews) is the second most expensive spice after

saffron. It is a tropical climbing orchid from the Orchidaceae family which consist

of 25,000 different species with nearly 110 species in vanilla genus. In the genus of

Vanilla, there is a large leaf species and also species without leaf (Medina, 2009). V.

planifolia is originated from Caribbean Islands, South and Central America. During

the Spanish conquest, vanilla was acquired from Aztecs and brought back to Europe

in 1510. Since then it had become a highly demanding spice required from all over

the world (Lubinsky et al., 2008; Kull et al., 2009; Odoux and Grisoni, 2011).

Vanilla planifolia is a perennial, and herbaceous plant with have thick

stemmed vine. All vanilla species produces flowers under optimum condition.

Vanilla plant produces 12 to 20 yellowish-green flowers in a cluster. The fruits of

the plants are similar to green bean in the form of a 6 to 10-inch-long pod with

thousands of minuscule black seeds inside each pod. This plant is a climber that has

tendril like roots and is able to grow up to 60 to 80 feet tall. In vanilla’s origin

habitat, the flowers are pollinated by melipona bees and hummingbirds. The flowers

will remain open for 24 hours and will wilt and drop off if they are not pollinated.

Hence, when the natural pollinators were absent in others part of the world for

vanilla flowers pollination, human hand-pollination become very essential in order to

get vanilla seed pods (Kull et al., 2009, Havkin-Frenkel and Belanger, 2011).

Vanilla plant is the only source that produces natural vanillin. Fresh vanilla

pods do not produce any flavour. The vanillin is bound and has to undergo a series

of processes to allow the vanillin to be released. Pure vanilla extract consists of

2

hundred different chemicals that is unable to be synthesized in laboratory. The

vanilla bean which is the sources of the vanillin is used in varies kind of food

industries including chocolates, ice-cream, soft drinks, liquors, sweets, cakes,

biscuits and others food products. In addition, vanilla also been used in the perfumes

and pharmaceuticals industries (Raghavan, 2006; Shinha et al., 2008; Kull et al.,

2009; Havkin-Frenkel and Belanger, 2011; Glass, 2011).

The vanilla plant requires specific soil and climate to grow. It is also very

susceptible to pests and diseases (Odoux and Grisoni, 2011). Vanilla plant is

conventionally propagated by using vegetative propagation such as stem cutting

technique. However, this method is slow and requires scarification of mature plant

in order to get the stem cut. Due to these disadvantages, micropropagagtion

technique was introduced as a better solution improves the cultivation of vanilla.

This method had been proven to be more efficient than conventional vegetative

propagation method which ensures constant high multiplication rate with sterile and

disease free planting materials (Havkin-Frenkel and Belanger, 2011).

Medium formulation plays an important role for optimum multiplication of

explants in micropropagation technique. The basal culture medium that commonly

use is MS medium, formulated by Murashige and Skoog (1962). Plant growth

regulators are essential under in vitro sterile environments where they act as a

stimulator for the initiation of adventitious shoots, roots formation or even callus

formation. For example, cytokinin is used for induction of multiple shoot formation.

Cytokinin is able to stimulate axillary shoot formation. However, in high

concentration, it will inhibit the root formation and even retard the plant from

maturing. Indeed, a specific concentration of plant growth regulator provided to the

plants is essential for enhance of growth efficiency (George et al., 2008).

3

Different organic additives also influence the growth of the plantlets. Some

organic additives can be used as natural plant growth regulator, extra nutrient

provider, anti-oxidant or even growth promoter. The commonly used organic

additives included banana homogenate, charcoal and coconut water for enhancement

of growth or absorption of phenolic compound which enhance the growth efficiency

(George et al., 2008). However, there are plenty of organic additives that potentially

benefit to the plants growth and development.

Histological techniques are method to study the anatomy of selected sample

which has been widely used in many research areas (Dolphin, 2001; Jain and Gupta,

2005). The major histological studies were focus on the vanilla beans instead of the

plant itself (Odoux et al., 2006; Mariezcurrena et al., 2008; Nishimura and Yukawa,

2010). Palama et al. (2010) had conducted study on histological study on shoot

differentiation from protocorm callus cultures of V. planifolia. However, it did not

cover the other plant parts that are vital for the plant survival in the natural

environment.

Hence, this study provides the opportunity to assess the effect of organic

additive as an alternative additive for enhancement of plant growth by using V.

planifolia as a model which is important by providing sustainable and cost efficient

method for enhancement of plant growth. This study also provides the opportunity

to understand the morphological, anatomical and physiological information of V.

planifolia plant adaptation from in vitro environment to the ex vitro environment.

This is vital for understanding the effect of micropropagation factors that cause

impact on the plant growth and development and also provide the information for

enhancement of micropropagation method in the future.

4

1.1 Objectives

The objectives of this study are:

i. To induce multiple shoot formation of Vanilla planifolia Andrews and

optimise the plantlets growth under in vitro condition,

ii. To determine the effect of different organic additives towards

improvement of the number of the vanilla plantlets,

iii. To establish acclimatization protocol for vanilla plantlets,

iv. To study the histological difference between in vitro plantlets and in vivo

plants.

5

CHAPTER 2

LITERATURE REVIEW

2.1 Orchids

Orchids are monocotyledon plants under the Orchidaceae family which also

the largest and most evolved within the plant family. They make up to 10 percent of

the flowering plants and consisted of about 30000 species and 850 genera. They

colonized almost every country except true desert and frozen Antartica. Orchids are

well known for its ornamental value because of its exotic, complex beauty flowers

(Arditti, 2008). At present, orchids have become million-dollar business, primary

due to floriculture cut flowers and potted plants trades (Roberts and Dixon, 2008;

Chugh et al., 2009; Hossain, 2011). Orchids are not only grown as ornamentals

plants; some also are used as medicinal herbs. Many species belong to genera of

Anoctochilus and Dendrobium has been reported to have medicinal properties and

they are commonly used in Chinese traditional folk medicines (Pant, 2013).

2.1.1 Vanilla genus

Vanilla genus consists of about 110 species belongs to the orchid family

which ranges from tropical to sub tropic regions. Tropical America has the most

diverse species of vanilla followed by south-east Asia and New Guinea. Vanilla

genus is a vine-like plant with climbing characteristic. They can grow up to 35 m

with alternate leaves along the stems. Vanilla leaf is commonly oblong in shape and

dark green in colour. However, there are some species that their leaves have reduced

to scales or totally leafless. Aerial roots grow from each node to allow the plant to

climb (Bory et al., 2008; Kull et al., 2009).

6

2.1.2 Vanilla planifolia Andrews’ history and distribution

Vanilla planifolia is native to Mexico and is cultivated in almost every

tropical region all over the world for the production of vanilla pod. Vanilla was first

used by the Aztecs to flavour their chocolate drinks. The Spanish brought back the

vanilla in 1510 and introduced to Europe (Ramachandra Rao and Ravishankar 2000;

Havkin-Frenkel and Belanger, 2007; Odoux and Grisoni, 2011).

The vanilla plant is a perennial herbaceous climber plants with thick stemmed

vine (2n=32). The plant produces 12 to 20 yellowish-green flowers in a cluster. The

fruit capsules which look like green bean were produced after pollination of the

flowers. The thick tendril-like roots opposite each leaf enable the plant to climb. The

wild types can even climb up 60 to 80-foot-tall which makes them the tallest of all

orchids. The roots can also be used for absorbing water and nutrients. The cluster

flowers open one at a time for twenty-four hours before the flowers drop off. Hand

pollination must carry out in the morning for fertilization. The beans are collected

before it completely matures. The beans are then undergoing a series process of

curing for 3 to 6 months before shipped for extraction (Havkin-Frenkel and Belanger,

2007; Lubinsky, 2008; Kull et al., 2009).

Vanilla production can be affected to climate and rainfall. The average

temperature for the vanilla plant growth is about 26 ± 4℃. Warm, moist, tropical

climates with averages 200 cm annual rainfall is the suitable condition for the

plantation of vanilla. However, vanilla can have root rot and others disease problems

due to excessive rain and long duration of drying section can kill the plant. Hence,

for the cultivation of vanilla, soils that can prevent water logging should be applied

(Havkin-Frenkel and Belanger, 2007; Odoux and Grisoni, 2011).

7

The bean extraction, the complex vanillin compound is used to flavour

chocolate, cakes, cookies and ice cream. Vanillin is also used as a fragrance for

perfume, hand lotion, shampoo and room freshener (Augstburger et al., 2000).

Vanilla was also added into the recipe of Coca-cola as one of the essential

ingredients of inverted by Atlanta chemist John S. Pemberton which went on sale in

1886 (as been cited by Glass, 2011). Besides that, it also been used in the perfumes

and pharmaceuticals industries (Kull et al., 2009; Havkin-Frenkel and Belanger,

2011).

Vanilla bean was an expensive vanillin sources toward the end of the 50’s

until it replaced by synthetic vanillin. Synthetic vanillin is cheaper compare to

natural vanillin and it sold 15000 metric tons per year with the price 15 $/kg compare

to nature vanillin that produce around 2000 tons annually with 1200 $/kg (Gallage

and Møller, 2015).

2.1.3 Vanillin and its uses

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the major component in

cured vanilla pods that is used as flavouring. Only 1%-2.5% of the vanillin is

obtained from a cured vanilla pods. Vanillin is white crystalline powders which

belong to the C6-C1 phenolic compounds as shown at the Figure 2.1 (Shinha et al.,

2007; 2008).

Chemical derived vanillin has serious issue where the usage of poison or

hazardous chemicals for purification of vanillin which could cause pollution to the

environment. Hence, the researches for the environmental friendly pathway for

producing synthetic vanillin have been continuing expanding (Dignum et al., 2002;

8

Havkin-Frenkel and Belanger, 2011; Gallaga and Møller, 2015). Due to its desirable

flavour, vanillin has been commonly used as primary flavour or as a component of

another flavour in the industry. The products that used vanillin as flavouring agent

include household products (cleansers, laundry detergent, and dish washing liquid),

dairy food products (ice cream, yogurt, flavoured milk, coffee creamers, chocolate

and etc.), baked goods (cupcakes, breads, cakes and etc.), beverages (carbonated

drinks, coffee, chocolate drinks, alcoholic drinks and etc.), pet products,

pharmaceutical products, perfume, and even toys (Havkin-Frenkel and Belanger,

2011).

9

Figure 2.1: Vanillin compound structure

10

2.2 Plant tissue culture system

Micropropagation is an in vitro asexual propagation using pieces of selected

plant called explants which can be cells, tissues, or organs isolated from the mother

plant cultured on an aseptic culture medium. In micropropagation, explants mainly

derived from shoot tips, axillary buds, stem sections, leaf sections or seeds. The

explant will be placed into the sterile nutrient medium which provides the essential

nutrients for plant growth and differentiation. The plant growth will be monitored

under control environmental condition with specific illumination, temperature and

humidity (Thorpe, 2007; George et al., 2008).

Plant tissue cultures enable studies to be conducted in controlled

environment without seasonal affection. There are four major stage of plant tissue

culture. The first one stage is establishment. The plant parts or explants from the

selected plants were taken from in vivo condition into sterile in vitro condition. The

explant will be surface sterilized accordingly before placed into the growth medium.

The second stage is multiplication. The sterile explant from the first stage will be

multiplied by using different protocol. Usually, plant growth regulators will be used

to ensure the explant to produce more plantlets, calli or cells. The third stage was

pre-transplant where it involves the plantlets which produce from the in vitro

condition undergo a series of hardening process in order to survive the natural

growth environment. The last stage is the acclimatization or transfer from culture to

the in vivo condition or the natural growth environment (Arditti, 2008; George et al.,

2008; Kärkönen et al., 2011).

11

2.2.1 Orchid micropropagation

Plant tissue culture technique has been accepted as a breakthrough for orchid

propagation, especially for the purpose of conservation of endangered orchids. This

technique has used to conserve many species of wild orchids, subsequently reduces

the number of collection form the wild. Many economically important orchids grow,

develop protocorms and propagate slowly naturally. This led to the development of

orchid tissue culture to enhance the productivity to fulfil the demand of orchid as cut

flower, pot flower and even medicinal purpose (Arditti, 2008; Kull et al., 2009; Deb

and Pongener, 2012; da Silva, 2013; Pant, 2013).

Development of different plant parts as explant for micropropagation of

orchids is very essential. The selection of explant plays an important role for the

succession of micropropagation of orchids. Shoot tips have been reported to be

effectively used for induction of shoot and protocorm-like bodies (PLBs) of many

orchids. Seeni and Latha (2000) had been reported the use of shoot tip culture

technique for successful establishment and multiplication of Vanda coerulea from

the forest of Western Ghats. Geetha and Shetty (2000) reported the development of a

large-scale micropagation protocol for Vanilla planifolia using both shoot tips and

nodal buds. Accordingly, they used two types of media, the establishment medium -

MS + 1 mg/L BAP + 3% sucrose and the multiplication medium - N69 (Nitsch basal

medium; Nitsch, 1969) + 0.5 mg/L BAP + 0.5 mg/L biotin + 0.5 mg/L folic acid and

2% sucrose. According to their report, these two medium were able to produce up to

100,000 plants in about 15 subcultures from single explant. However, Kalimuthu et

al. (2006) reported that by using a single type of medium using MS + 1 mg/L BAP +

150 mg/L of coconut water for initiation, multiplication, elongation and rooting of

Vanilla. Gonzalez-Arnao et al., (2009) had reported successfully develop in vitro

12

fragmented explants (IFEs) technique by using the remaining base of the vanilla

plant clusters as explant producing up 15 plantlets after 4 months of culture in ½ MS

medium supplemented with 1 mg/L of BAP, and 0.5 mg/L of indole-3-butyric acid

(IBA).

2.2.2 Culture medium

Naturally, plants obtained nutrients from the soil. Culture medium is a source

of nutrients designed to provide necessary nutrients to the plant cells to grow. Indeed,

culture medium is one of the most important factors for in vitro culture technique is

the plant culture medium. Plant propagation is greatly influenced by the formulation

of the culture medium used for a successful micropropagation (George et al., 2008;

Kärkönen et al., 2011). In vitro culture media are generally made up from these

components: macronutrients, micronutrients, vitamins, amino acids or other nitrogen

compounds, sugar, other organic compounds, solidifying agents, and growth

regulators. For orchid’s plant tissue culture purpose, the common micropropagation

mediums are MS medium [1962], Vacin and Went medium [1949] and Knudson C

medium [1946] (Murashige and Skoog, 1962; Arditti, 2008; George et al., 2008;

Pant, 2013).

Carbon source in the form of carbohydrates are supplied to the in vitro

plantlets as a source of carbon dioxide and osmotic agent. The carbon source is

supplied to the plant for assimilation of CO2 during photosynthesis process. Hence,

in vitro plants depend on the supplied carbohydrates for energy supply in order for

the plant to grow. There are many carbohydrates available to supply into culture

medium, however sucrose is the most common used as it is cheap and easily obtained

(Caponetti et al., 2000; Arditti, 2008; George et al., 2008). This carbon source

13

required for plant growth was varying according to plant species. Sucrose

concentration at 65 g/L produced optimum level in-term of multiplication rate of

plantlets, and root numbers in date palm (Abdulwahed, 2013).

Plant’s growth required large amount of macronutrients and comprise at least

0.1 % of the dry weight of plants are included ions of nitrogen (N), potassium (K),

calcium (Ca), phosphorus (P), magnesium (Mg) and sulphur (S) (Caponetti et al.,

2000; George et al., 2008). These macronutrients are required as major component

of amino acids, plant energy metabolism, cofactor for plant enzymes, element in

osmotic potential of cells, component of cell walls, component of chlorophyll, and

etc. Lacking of these elements could cause stunted plant growth and unhealthy plant

growth. These nutrients are usually required in higher amount compared to

micronutrients (Mezzetti et al., 1991; Caponetti et al., 2000; Arditti, 2008; George et

al., 2008; Taiz and Zeiger, 2010).

The trace elements or micronutrients which are needed for small amounts and

necessary for plant growth and developments are included iron (Fe), nickel (Ni),

chlorine (Cl), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum

(Mo). They act as element in photosynthesis, break down urea, activation agent for

enzymes, component of alcohol dehydrogenase, and even responsible in cell

elongation, nucleic acid metabolism, hormone responses, and membrane function

(Mezzetti et al., 1991; George et al., 2008 Taiz and Zeiger, 2010).

Plants absorb these inorganic nutrients in ions form. The medium is

supplemented with these inorganic nutrients as salts that would dissolve in the

medium to form into cations and anions. Normal plants produce their own vitamins

as catalysts in different metabolisms. However, plant cells and tissues that are grown

14

in vitro will have difficulty of producing their own vitamins. Hence, vitamins such

as thiamine (B1), nicotinic acid (niacin) and pyridoxine (B6) are added to the culture

medium. Amino acids are a source of nitrogen that is rapidly taken up by the cells

for stimulation of cell growth. Amino acids are more easily absorbed by the plant

compare to inorganic nitrogen ion sources. The most commonly used amino acids in

culture medium are casein hydrolysate, L-glutamine, L-asparagine, and adenine

(George et al., 2008 Taiz and Zeiger, 2010).

2.2.3 Gelling agent

The growth of shoots or roots is greatly affected by the physical stability of

the culture medium. The solidification of the medium has acts as a support system to

ensure the plant tissues and organs to stay above the nutrient medium as well as

supporting the plant root growth. Many gelling agents are used for plant culture

media, e.g., agar, agarose, phytagel and Gelrite (George et al., 2008). There are

various brands and grades of agar, with different price, and composition. The choice

of agar band mostly determines by actual use and experience for a plant species. For

industrial scale micropropagation, cheaper brands of agar are more economically

practical where higher grade agar is unnecessary (Prakash et al., 2004; George et al.,

2008). Mengasha et al., (2012) has reported to use enset starch as an alternative

gelling agent for Vanilla planifolia culture medium. This enset starch has reported

have low charity that can affect contamination detection; however, it also reported

that it able to enhance root proliferation. Zimmerman et al. (1995) had reported using

corn starch gelled medium for tissue culture of six cultivars of apple together with

two red raspberries. Compared to MS medium gelled with agar (7.5 g/L), the fruit

15

crops were reported to have equal or better shoot proliferation on MS medium gelled

with a mixture of corn starch (50 g/L) and Gelrite (0.5 g/L). However, the medium

appeared to be gray-white in colour causing the difficulty for the detection of

contamination.

Agar as a gelling agent remains the popular choice for solidification of plant

culture media since 25 years ago. It has been used for solidified the culture medium

used for higher plants. However, it had been reported that agar may cause growth

inhibition in plants (Prakash et al., 2004). Scholten and Pierik (1998) even reported

within different brand of agars affected major differences in the grow rate of plantlets.

Gelrite has become the preferred gelling agent after the agars due to its purity

and consistent quality. Relatively smaller quantities of Gelrite able to produce the

same gelling property as compare to agar (Harris, 1985). The cucumber somatic

embryos had higher germination rate when the culture media were solidified with

Gelrite (Ladyman and Girard, 1992). However, Gelrite had also been reported that

could cause hyperhydricity in Aloe polyphylla as compared to agar-solidified

medium (Ivanova and Van Staden, 2011).

‘Isabgol’, the Plantago ovata seeds’ husk had been reported to be used as a

gelling agent in tissue culture medium. The large quantity of mucilage present in the

husk is colloidal and polysaccharidic which is able to soluble in hot water, forming

viscous liquid and sets to gel when cooled. The medium can be gelled with 30 g/L of

‘Isabgol’. The plant growth on media gelled with ‘Isubgol’ were no significant

difference to that on media gelled with agar (Babbar and Jain, 1998). Agrawal et al.

(2010) in another hand used 35g/L of ‘Isabgol’ to solidified the medium for in vitro

micropropagation of banana.

16

2.2.4 Cytokinin

Cytokinins are growth regulators that are used for stimulating cell division

and other plant growth. It is the most commonly used hormone or plant growth

regulators for stimulating the respond of multiple shoot formation in plant tissue

culture. Cytokinins that widely used nowadays are 6-benzylaminopurine or 6-

benzyladenine (BAP, BA), 6-γ-γ-dimethylaminopurine (2iP), N-(2-furanylmethyl)-

1H-puring-6-amine (kinetin), and 6-(4-hydroxy-3-mehty-trans-2-butenylamino)

purine (zeatin) (George et al., 2008).

Cytokinins were able to promote proliferation of shoots and inhibit shoot

elongation. Elongation of Arabidopsis thaliana was retarded after sprayed with 22.5

mg/L BAP (Greenboim-Wainberg et al., 2005). Higher concentrations of cytokinins

caused abnormal growth where too many small shoot is produced and fail to elongate

eventually. Higher level of cytokinins may cause the leaves of some species became

abnormal in shape and the shoot will become hyperhydricity (George et al., 2008).

2.2.5 Organic additives

Organic additives are undefined composition materials which can be used for

establishment of the cultures. These organic additives may act as extra carbon

sources, nitrogen sources, and even as plant growth regulators. These organic

additives included coconut water (CW), orange juices, banana pulp, tomato juice,

charcoal, potato extract, yeast extract and malt extract (Agarwal et al., 2004; Aktar,

2008; Daud et al., 2011). These organic additives had been used to enhance plant

growth, PLBs regeneration, PLBs proliferation, enhance callus growth and etc

(Rahman et al., 2004; Aktar et al., 2008; George, 2008). These complex additives

17

can be very effective in enhances the in vitro plant growth of orchid seedling with its

undefined organic nutrients and growth factors. Tharapan et al. (2014) reported that

potato extract able to enhances the shoots growth of Dendrobium orchids which soy

milk able to enhance the roots growth. These organic additives were simple,

efficient, effective, and convenient to enhance culture media for commercial

production. Optimization and adjustment are required for optimum growth of the

culture. These enhancement effects of the organic additives work differently on

different plant. Some organic additives were reported enhances plant growth but

some were reported inhibit plant growth (Arditti, 2008).

2.2.5.1 Coconut water (CW)

Coconut plant (Cocos nucifera L.) has long been recognized an important

crop for human life. The coconut water (CW) which is the liquid endosperm has

assumed the most nutritious beverage provided by the nature (Khan et al., 2003). In

plant tissue culture, coconut water is a common organic additive that has long been

used as a growth promoting component which contained many uncharacterized

biochemical compounds than directly or indirectly enhances the plant growth rate.

Coconut water can induce plant cells to develop and differentiate rapidly. According

to Ge et al. (2004; 2005; 2006), they had identified few types of natural occurrence

cytokins present in coconut water which includes zeatin, zeatin riboside, trans-zetin,

O-glucoside, dihydrozeatin O-glucoside, ortho-topolin, kinetin free base and kinetin

riboside. Coconut water had reported containing natural indole acetic acid (IAA)

which used to induce adventitious rooting of Dracaena purplecompacta L.

(Agampodi and Jayawardena, 2009). The overall phosphorus content of the media

18

was reported to be increased when coconut water was supplemented (Mezetti et

al.,1991).

2.2.5.2 Coconut sugar

Coconut sugar is made from the sap from coconut tree (Cocos nucifera L.)

blossoms. Generally, the sap collected will be boiled to produce brown solid sugar

block. This sugar is widely used in Malaysian’s foods and drinks preparation

(Apriyantono et al. 2002). It has been reported that commercial sugars had total

sugar content between 761.96 – 841.23 mg/g dry weight with 0.15-122.07 mg/g of

reducing sugar content. Thus, comparing to commercial sugars, it have been

reported that coconut sugar has lower total sugar content but higher in reducing sugar

content. According to Kongkaew et al. (2014), some amount of calcium, iron,

sodium, magnesium, and potassium were detected. They were found slightly higher

compared to commercial sugar. Besides that, it has also found to have slightly higher

antioxidant activity compared to commercial sugars (Kongkaew et al, 2014). It had

been reported that sucrose can be replaced by 6% date syrup for culturing the in vitro

somatic embryogenesis of date palm (Alkhateeb, 2008).

2.2.5.3 Activated charcoal

Activated charcoal has been widely used in many industrial due to its ability

to absorb and removal of the harmful or unwanted substances which included food,

beverage, pharmaceutical, chemical and even biotechnology industries. It has been

widely used to treat drinking water and wastewater which had been proven to have

19

the ability to absorb organic pollutant (Elhussien and Isa, 2015). Activated charcoal

has included animal and plant charcoal, but plant charcoal is preferable in plant

tissue culture as additive in the medium to enhance the growth performance such as

seed germination, somatic embryogenesis, protoplast culture and others

micropropagation technique (Thomas, 2008).

For in vitro micropropagation, it is very common for the plant to exudate its

phenol compound and it often affect the plant growth. Polyphenols released by

Aristolochia indica had reported affecting the shoot growth when culture in in vitro

condition (Soniya and Sujitha, 2006). Many in vitro explants would have explant

browning problem. In order to encounter this type of situation, activated charcoal

was added into the media to avoid the explant browning (Wang et al., 2005; Feyissa

et al., 2005). In addition, activated charcoal combined with TDZ supplemented

medium also able to promote and enhance the multiple shoot formation of the

seedling of Rhynchostylis retusa (Thomas and Michael, 2007).

2.2.5.4 Pineapple juice

Pineapple (Ananas comosis) is widely grown in tropical countries. As being

one of the organic additives, it was not being use as frequent as coconut water.

Pineapple fruits, the edible portion, contain about 85% water, 0.4% protein, 14%

sugar, 0.1% fat and 0.5% fibre. In the total 14% of sugars content – half of these is

sucrose with another half of glucose and fructose. It is also rich in calcium,

phosphorus, iron, calcium, vitamins A and B together with 7-9% of citric acid

(Vaughan and Judd, 2006; Pua and Davey, 2007, Patil et al., 2011). The growth

promoting activity of the pineapple is still yet to be completely understood.

20

Santiago-Silva (2011) reported that detection of several polyamines in the pineapple

fruits. Wisziewska et al. (2013) confirmed that pineapple pulp was able to promote

root development in Daphne plants. They suggested that this might due to the

presence of rooting cofactors and polyamines stimulates formation of the rooting.

2.2.5.5 Dragon fruit (pitaya) juice

Dragon fruits/pitayas (Hylocereus spp.) are perennial climbing cactus origin

from tropical regions of America. It has been commercially available in Malaysia.

However, due to its new to the market, information from the research conducted was

limited (Lim et al., 2007).

The fresh fruit contains about 86% of water, with 5-8% of total sugars

content. These sugars content mainly derived from reducing sugar such as glucose

and fructose where sucrose was only found about 3-8% of total sugars (Le Bellec,

2006). Charoensiri et al. (2009) reported that dragon fruits content some amount of

beta-carotene, lycopene, and vitamin E. According to Lim et al. (2006), dragon fruit

has relative low total phenol compound and ascorbic acid content compared to others

Malaysia’s local fruits. Dragon fruit contain high proline content which range from

1.1 to 1.6 g/L of juice and proline is well-known of helping plant species to adapt to

the environment stress. It also contains high amount of potassium, magnesium and

calcium (Le Bellec, 2006, Szabados and Savoure ,́ 2009).

21

2.2.5.6 Banana pulp

Bananas (Musa spp.) are common fruits that grown throughout the tropical

countries. In 100g of fresh fruits, banana contained about 15g of starch and 12 g of

total sugars. It also contains various vitamin and trace minerals particularly high

amount of potassium, calcium, phosphorus and also tryptophan that can promote the

plant growth (Sharaf et al.1979; Gnasekaran et al., 2010; USDA, 2015). Addition of

banana into the culture medium for orchid growth became widely used to enhance

growth of the plants. Due to the dark colouration, banana additive culture media are

easily recognized. It has been reported that ripe banana pulp is able to stimulate the

growth of vanilla seedling and immature embryos (Withner, 1955). However, the

mechanisms of the enhancement of banana towards the in vitro plantlets are not clear

(Arditti, 2008). It has been reported that banana could stabilize the pH when

supplemented into the medium. However, high concentration of banana pulp can act

as inhibitor to the plants growth (Gnasekaran et al., 2010).

According to Aktar et al. (2008), 10% w/v of Sabri banana pulp

supplemented into ½ MS medium had significantly enhance the growth and

development of Dendrobium orchid PLBs. PLBs proliferation of Phalaenopsis

violacea orchid showed 10% increment with Pisang Mas (AA) pulp (Gnasekaran et

al., 2010). Obsuwan and Thepsithar (2014) reported that Vanda Tokyo Blue

seedling cultured in Vacin and Went (VW) medium supplemented with banana pulp

showed increased in fresh weight and root number. However, Nambiar et al. (2012)

reported that banana pulp did not promote the proliferation of PLBs in Dendrobium

Alya Pink.

22

2.2.5.7 Green tea leaf extract

Tea is one of the most popular drinks in the world. Green tea is prepared by

drying the fresh tea lead without fermentation. Green tea is rich in catechins, one of

the flavanol which make up of 30% of dry leaf weight. There are also others

flavonols compounds and their glycosides. Total caffeine content is about 3% of dry

leaf weight. Tea leaf also is rich in potassium, calcium, magnesium and aluminum.

Tea leaf has high antioxidant property due to its various flavonoids compound and

bind with others molecules even proteins to form non-toxic component. Hence, it

also reported to have the ability to inhibit enzymatic activity. It can also stimulate

the modulation of receptors, enzymes, or regulatory proteins at low concentration

(Graham, 1992; Balentine, 1997; Rani et al., 2014). Jeszka-Skowron and Zgola-

Grzekowial (2014) reported the optimized protocol for extraction green tea

compound by infusing the green tea leaves in 95℃ for 15 minutes. In animal tissue

culture, tea leaf extract had been reported added into the tissue culture medium.

Barakat et al. (2014) reported that 0.3 g/L of green tea leaves extracts able to

enhance the sheep blastocyst formation. Green tea polyphenols also reported to be

used in enhancement of cow oocytes development (Wang et al. 2012).

2.2.6 Rooting

Rooting of in vitro plantlets is one of the important stages in

micropropagation system (Thiart, 2003). Plantlets grow inside in vitro environment

are subsequently small and fragile where survival ex vitro will be typically difficult

(Thiart, 2003). Rooting of plantlets ensure the plantlets develop the ability to obtain

nutrients from the soil efficiently (Thiart, 2003). This ensures the plants to be able to

23

survive outside the sterile environment. Different plants required different conditions

for rooting induction, hence different formulations of in vitro culture medium is

require for different type of plantlets to establish their rooting (George et al., 2008).

Lee-Espinosa et al. (2008) reported that V.planifolia able to promote rooting when

culture in ½ MS media supplemented with 0.44 µM naphthalene acetic acid (NAA).

However, Zuraida et al. (2013) reported V. planifolia able to produce 100% rooting

when culture with ½ MS without any plant growth regulators.

2.2.7 Acclimatization

Acclimatization is the method which in vitro plantlets were transferred from

in vitro condition to the greenhouse condition. High humidity and a low light

intensity of the in vitro condition caused the plantlets to be very fragile. The stomata

of the leaves may fail to close under low humidity conditions such as green house or

field condition. The plantlets lose water rapidly through transpiration when they are

moved out from in vitro environment. The plantlets would not be able to absorb

enough water for the transpiration process. This eventually causes the plantlets to

wilt. However, once the plants are slowly adapting the external condition for couples

of few days, the plants will be able to survive in the green house or field. To

facilitate this, the plantlets are kept in a place with high humidity and low light

intensity for several days before transfer to greenhouse condition (George et al.,

2008).

Parthasarathy and Nagaraju (1999) reported the use of jars as acclimatization

container for rooted Gerberawas found to be better than pots as jars able to maintain

high humidity around the plant and reduce the chance for the plants to be dry out

which increased the survival rates before transfer to the field. There are numerous

24

types of potting mediums which can be used in acclimatization such as peat, sand,

perlite, soilrite and others soil mixtures. Sherif et al (2012) had used coconut coir,

activated charcoal and commercial fertilizers in the ratio of 3:1:1 to acclimatize the

Anoectochilus elatus Lindley in greenhouse. The rooted vanilla plantlets were

transferred to hardening medium with top soil and compost mixture at the ratio of 2:1.

The survival rate of the plantlets achieved 90% of total plantlets after 4 weeks’

culture in glasshouse condition (Zuraida et al., 2013).

Peat or sphagnum moss were commonly used as orchid potting material due

to its characteristics of spongy fibrous texture, a high porosity with high water-

retaining and low ash content (Zettler et al., 2007; Dutra et al., 2008; Bunt, 2012). V.

planifolia also reported to be acclimatized by potting in peat moss and agrolite (1:1)

in the green house condition for 30 days with 90% survival rate before transplanted

to soil in the field (Lee-Espinosa et al., 2008).

2.3 Histological analysis

In order to understand the inner and outer structure changes of the plants

which undergo the acclimatization process, histological studies such as light

microscopy study and scanning electron microscope (SEM) analysis should be

carried out. These studies enable researcher to understand the microstructure

changes and inner cellular changes of the plants from in vitro condition to ex vitro

condition.

Histological changes occur due to the cellular process can be studied and

provides information that allow further research study which improved the

understanding of normal and abnormal functions of the plant (Yeung, 1998; Jain and

Gupta, 2005). Niranjan et al. (2009) suggested that by studying the basic

effects of selected additives for growth efficiency...

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